This project focuses on the molecular mechanisms that mediate programmed cell death (PCD) in plants, specifically in response to pathogen infection. We have previously shown that loss-of-function mutations in the EDR1 gene of Arabidopsis confer enhanced resistance to infection by the powdery mildew fungus Erysiphe cichoracearum. Significantly, this resistance is correlated with a more rapid activation of several defense responses, including PCD, indicating that EDR1 is a negative regulator of PCD. We have also shown that edrl mutants have enhanced sensitivity to the plant hormone abscisic acid (ABA) and that they senesce more rapidly than wild-type plants. EDR1 thus represents an excellent entree into understanding how induction of PCD is regulated at a molecular level during pathogen infection and senescence. Work during the first three years of this project has identified two likely substrates of EDR1, a kinesin-like protein known as KCA1and an ABA-inducible transcription factor called AtMYC2. We have also identified a large number of mutations that suppress ec/rf-mediated resistance. Finally, we have identified two additional genes that confer ec/rf-like resistance phenotypes when mutated: EDR2, which encodes a novel protein containing two lipid-binding domains, and DRP1E, which encodes a dynamin-related protein. Both EDR2 and DRP1E appear to be localized to mitochondria, providing a mechanistic link between mitochondria and regulation of PCD in plants. Our specific aims in this competing renewal are to 1) clone the genes identified in our suppressor screen;2) determine whether EDR1 phosphorylates AtMYC2 and KCA1in vivo, and if so, how phosphorylation affects their function and stability;and 3) determine the lipid binding properties of EDR2 and DRP1Eand assess how lipid binding affects powdery mildew-induced PCD and mitochondria! function. Specific aim 1will be accomplished by standard positional cloning methods. Completion of this aim will uncover additional genes in the EDR1 pathway,as well as reveal other pathwaysthat interact with EDRL Specific aim 2 will be accomplished by monitoring charge differences on AtMYC2 and KCA1 in wild-type versus edrl mutant backgrounds, localizing GFPfusions in transgenic plants, and by double mutant analysis. Specific aim 3 will be accomplished using a protein-lipid overlay assay and a lipid vesicle tubulation assay with wild-type and mutant derivatives of EDR2 and DRP1E. Mitochondria! function will be assessed using a fluorescent dye that reports mitochondria! membrane potential. Together, these analyses will provide significant new insight into how pathogen-induced PCD is regulated in plants. This work will also shed light on mechanisms of pathogen defense and PCD in humans, as several of the proteins identified in this study share significant similarity with human proteins, including some linked to PCD and mitochondria! function in human cells.